WO2023140691A1 - Formulation orale contenant de la 5-aza-4'-thio-2'-désoxycytidine et son procédé de préparation - Google Patents

Formulation orale contenant de la 5-aza-4'-thio-2'-désoxycytidine et son procédé de préparation Download PDF

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WO2023140691A1
WO2023140691A1 PCT/KR2023/001028 KR2023001028W WO2023140691A1 WO 2023140691 A1 WO2023140691 A1 WO 2023140691A1 KR 2023001028 W KR2023001028 W KR 2023001028W WO 2023140691 A1 WO2023140691 A1 WO 2023140691A1
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aza
dcyd
drug
oral dosage
dosage form
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이진수
정두영
조현용
최신혜
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주식회사 피노바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an oral dosage form containing 5-aza-4'-thio-2'-deoxycytidine and a preparation method thereof.
  • Decitabine also called Dacogen® or 5-aza-2'-deoxycytidine
  • Decitabine functions by incorporating into DNA strands during replication, and when DNA methyltransferases (DNMTs), such as DNMT1, bind DNA and replicate methylation to daughter strands, DNMTs bind irreversibly to decitabine and cannot be separated.
  • DNMTs DNA methyltransferases
  • decitabine action is dependent on cell division. Cells must divide for the drug to work. Therefore, cells that divide much faster than most other cells in the body (such as cancer cells) are more affected by decitabine. That is, decitabine is used in the treatment of cancers such as leukemias, including myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML), in which DNA hypermethylation is important for development.
  • MDS myelodysplastic syndrome
  • AML acute myelogenous leukemia
  • aza-T-dCyd 5-aza-4'-thio-2'-deoxycytidine
  • NCI National Cancer Institute
  • This DNMT1 inhibitor has recently attracted attention due to its high DNMT elimination and cell inhibitory activity, reduced rate of cytidine deaminase degradation, and relatively low production of toxic by-products compared to existing compounds with a 5-azacytidine backbone.
  • aza-T-dCyd can be manufactured in a variety of forms and crystal structures.
  • U.S. Patent No. 5,591,722 relates to 2'-deoxy-4'-thioribonucleosides and intermediates useful for treating viral diseases and describes a general formula comprising 5-azacytidine compounds.
  • US Patent Publication No. 2006/0014949 reports polymorphs of decitabine.
  • Thottassery, et al. (Cancer Chemother Pharmacol, 2014) reports aza-T-dCyd.
  • Clinical trial NCT04167917 reports a phase I trial of Aza-T-dCyd in MDS and AML expected to be completed in 2025.
  • the polymorph of aza-T-dCyd has so far remained elusive.
  • DNA methyltransferase (DNMT) inhibitors based on cytidine analogs such as decitabine and azacitidine have excellent efficacy in the treatment of elderly patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • DNMT DNA methyltransferase
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • DNMT DNA methyltransferase
  • anticancer drugs of the cytidine analogue family such as decitabine or azacytidine, commonly have difficulties in being developed as oral anticancer drugs due to individual differences in absorption and metabolism rates in the human body.
  • the optimal dose for one individual is a dose that cannot show a therapeutic effect for another individual.
  • Gastrointestinal Tract refers to all organs of the digestive system from the mouth to the anus.
  • Absorption of a drug means the movement of substances into or across tissues, in particular, the movement of a drug to the wall of the gastrointestinal tract and then to the bloodstream.
  • Absolute bioavailability is the rate at which a drug is systemically utilized after oral, rectal, subcutaneous, transdermal, intranasal, or extravascular administration of the drug.
  • a dosage form is a means of controlling the action of a drug by adjusting variable factors affecting dissolution and absorption characteristics of a drug, which are drug-derived factors.
  • the physical state of the pharmaceutical raw material that is, whether it is crystalline or amorphous
  • the amorphous form has high solubility and is helpful in increasing the efficacy and showing the fast effect, but it is unstable and the shelf life is shortened, and it is also difficult to release the drug and control the blood concentration.
  • the crystalline form has low solubility and low bioavailability per unit weight, stability is secured and there is an advantage in making a continuous controlled release formulation, crystalline raw materials are used in most pharmaceutical formulations except for special cases.
  • the crystalline form of a drug affects the physical and chemical stability, hygroscopicity, and dissolution rate of a compound in water. Chemical instability causes restrictions on the manufacturing, management, transportation, and shelf life of pharmaceutical raw materials. It also affects the stability after formulation as an oral preparation. If the crystal form is different, the crystal shape, purity, yield, etc. are different, and it greatly affects the formulation process, manufacturing environment, and manufacturing cost.
  • the individual drug exposure of the aza-T-dCyd drug varies greatly due to the difference in the degree of absorption for each individual, so that the optimally designed dose for anticancer efficacy may be a dose that cannot show a therapeutic effect for each individual or a dose that causes serious toxicity.
  • an object of the present invention is to provide an oral dosage form in which the ratio (wt%) of crystalline Form A in aza-T-dCyd is adjusted to a known value within an acceptable error range, so as to calculate a single dose that exhibits a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug, and to minimize individual differences in absorption during oral administration to stably achieve the highest blood concentration (Cmax) of the aza-T-dCyd drug.
  • a first aspect of the present invention provides a method for preparing an oral dosage form containing aza-T-dCyd as an active ingredient, the efficacy of which is dependent on the highest blood concentration (Cmax), characterized by precisely designing a single dose that exhibits a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug within an acceptable error range.
  • a second aspect of the present invention provides an oral dosage form in which the ratio (wt%) of crystalline Form A in the aza-T-dCyd drug is controlled, wherein the crystalline raw material containing crystalline Form A in a desired ratio (wt%) is prepared from crude materials of aza-T-dCyd, which is a synthesized product of the aza-T-dCyd compound, and then formulated into an oral dosage form.
  • the exposure amount of the aza-T-dCyd drug in the blood can be stably implemented within an acceptable error range.
  • a third aspect of the present invention provides a method for preparing an oral dosage form containing aza-T-dCyd as an active ingredient, wherein the ratio (wt%) of crystalline Form A and/or crystalline Form F in the crystalline raw material of aza-T-dCyd is confirmed and then formulated into an oral dosage form.
  • a fourth aspect of the present invention provides an oral dosage form wherein a single dose of the aza-T-dCyd drug is designed such that the ratio of Form A is greater than or equal to the ratio of Form A corresponding to the inflection point of the Cmax phase at which the maximum blood concentration (Cmax) change value according to the change in the ratio of Form A in the same dose of the aza-T-dCyd drug is increased.
  • a fifth aspect of the present invention provides an oral dosage form containing at least 70% of Form A in the aza-T-dCyd drug.
  • a sixth aspect of the present invention provides an oral dosage form characterized by containing 30 mpk to 70 mpk of crystalline Form A when designing a single dose of the aza-T-dCyd drug.
  • a seventh aspect of the present invention is a method for preparing an oral dosage form containing an aza-T-dCyd drug whose efficacy is dependent on the maximum blood concentration (Cmax), comprising the steps of crystallizing the aza-T-dCyd compound in the presence of a solvent and then removing the solvent to convert it into a non-solvate crystalline form; and a second step of preparing an oral dosage form designed so that the non-solvate crystalline form prepared in the first step can be dissolved in the stomach.
  • Cmax maximum blood concentration
  • a drug is any substance used to diagnose, cure, alleviate, treat, or prevent disease (excluding food or devices) or to affect the structure or function of the body.
  • disease excluding food or devices
  • any chemical or biological substance that affects the body and its metabolism indicates the drug's atomic or molecular structure.
  • the aza-T-dCyd drug includes a compound represented by Formula 1 below as well as pharmaceutically acceptable salts thereof.
  • “Pharmaceutically acceptable salts” include non-toxic acid and base addition salts of the compounds to which the term refers.
  • solvate refers to a compound provided herein or a salt thereof that further comprises a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces.
  • the solvent is water, the solvate is a hydrate.
  • aza-T-dCyd of Chemical Formula 1 is a DNMT1 inhibitor based on a 4-Thio-2-deoxyribose backbone, and has both a sugar structure change (4'-thiodeoxyribose structure) and an aza-cytosine group.
  • aza-T-dCyd is activated by triphosphate in cells and used instead of some dC (deoxycytidine) during DNA synthesis. It is a nucleoside anticancer drug that induces cancer cell death by trapping DNMT1 after DNA synthesis and activating various epigenetic action mechanisms.
  • aza-T-dCyd due to its Thio-nucleoside structure, can exhibit strong anticancer efficacy by simultaneously blocking endonuclease that participates in DNA damage repair, an existing mechanism of resistance development, along with strong inhibition of DNMT1, the main drug target.
  • Aza-T-dCyd compounds can be rapidly activated by triphosphate in cancer cells compared to normal cells.
  • the Aza-T-dCyd compound delays DNA replication by causing base excision repair and/or mismatch repair through DNA insertion of aza-T-dCTP, a triphosphate of the aza-T-dCyd compound, thereby inducing replication stress and increasing DNA damage response.
  • the Aza-T-dCyd compound suppresses the expression of RRM1 protein, a ribonucleotide reductase that is important for dNTP de novo synthesis, induces DNA replication stress by reducing the amount of dCTP and dTTP in cells, and can generate a strong DNA damage response.
  • aza-T-dCyd significantly lowers the activation rate by dCK (deoxycytidine kinase) in normal cells due to its Thio-nucleoside structure, thereby selectively delivering the active ingredient of the drug to cancer cells.
  • dCK deoxycytidine kinase
  • Example 6 Furthermore, through the pharmacokinetic analysis of Example 6, (1) the anti-cancer treatment effect of aza-T-dCyd is Cmax dependent rather than AUC dependent; and (2) exposure to higher doses of the aza-T-dCyd drug for a short period of time is an effective anti-cancer therapy.
  • the present invention was completed based on these findings.
  • the optimally designed dose for anticancer efficacy may be a dose that cannot show a therapeutic effect or a dose that causes serious toxicity depending on the individual.
  • the proportion (wt%) of Form A in the drug aza-T-dCyd can be precisely controlled.
  • a single dose that exhibits a desired therapeutic effect is calculated from the highest blood concentration (Cmax) of the aza-T-dCyd drug, and the ratio (wt%) of Form A in the aza-T-dCyd drug is adjusted to a known value within an acceptable error range to stably exert the highest blood concentration (Cmax) of the aza-T-dCyd drug by minimizing individual differences in absorption during oral administration.
  • the method for preparing an oral dosage form containing aza-T-dCyd as an active ingredient according to the present invention is characterized by precisely designing a single dose that exhibits a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug within an acceptable error range.
  • "precisely designing a single dose that exhibits a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug within an acceptable error range” means, for example, designing a dosage form capable of controlling a constant dissolution rate profile and absorption of the drug within a predictable error range to minimize the variation in drug exposure, and based on this, a single administration of the aza-T-dCyd drug that achieves the highest blood concentration (Cmax) that exhibits the desired therapeutic effect It could be capacity design.
  • the pharmacokinetic parameters for evaluation of bioavailability are the amount of drug that the active ingredient or its active metabolites reach the systemic circulation from the preparation and the time it takes for them to reach the systemic circulation.
  • the area under the plasma level-time curve refers to the degree of drug exposure, that is, the degree of bioabsorption of the drug, and reflects the total amount of active drug that reaches the systemic circulation.
  • the unit of AUC is expressed as concentration ⁇ time (eg, ⁇ g hr/mL).
  • AUC is a measure of the degree of drug bioavailability. It represents the extent of total systemic exposure.
  • Cmax is the highest blood concentration after drug administration and is an indicator indicating whether the drug is sufficiently absorbed into the systemic circulation to show a therapeutic response, as well as providing information on whether or not it can cause toxic effects.
  • t max is the time at which the blood concentration reaches the highest value after drug administration, and refers to the moment at which the absorption rate of the drug and the excretion rate become the same as the time point at which the drug absorption reaches the highest value. After t max , drug absorption continues but slows down. Therefore, when comparing the absorption of drugs, it is an index for the rate of absorption.
  • IC 50 values are related to AUC. Usually, the dose can be calculated from AUC and bioavailability.
  • the present invention is characterized by designing a single dose of the aza-T-dCyd drug so as to exert a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug.
  • a single dose of the aza-T-dCyd drug may be 5-70 mg/m 2 .
  • it may be 5-10 mg/m 2 .
  • it may be preferably 5 to 55 mg/m 2 , more preferably about 5 to 30 mg/m 2 , and still more preferably 5 to 20 mg/m 2 .
  • in combination therapy it may be 5 to 10 mg/m 2 .
  • NOAEL No-observed-adverse-effect level
  • HNSTD highest non-severe toxic dose
  • the appropriate dosage for humans when calculating the appropriate dosage for humans, it can be adjusted to be less than the no-observed-adverse-effect level (NOAEL) or the highest non-severe toxic dose (HNSTD) that does not cause serious toxicity, and the dose of the anticancer agent can be compared with the non-toxic amount (NOAEL) or the highest dose that does not cause serious toxicity (HNSTD), and the risk factor (risk) during administration can be predicted.
  • NOAEL no-observed-adverse-effect level
  • HNSTD non-severe toxic dose
  • the peak blood concentration (Cmax) after administration of a drug is not only an indicator of whether a drug has been sufficiently absorbed into the systemic circulation to produce a therapeutic response, but also provides information on the toxicity profile.
  • PK pharmacokinetics
  • IV intravenous
  • PO oral
  • mpk milligram per kilogram
  • Example 11 when comparing the results of 2mpk treatment and 1mpk treatment of mice twice, in consideration of the fact that the weight loss of mice was severe in the latter, the present invention provides that the highest blood concentration (Cmax) of the aza-T-dCyd drug calculated from a single dose of the aza-T-dCyd drug is a non-toxic level (No-observed-adverse-effect level, NOAEL) or the highest that does not cause serious toxicity.
  • NOAEL No-observed-adverse-effect level
  • a single dose of the aza-T-dCyd drug can be designed to be lower than the value corresponding to the highest non-severe toxic dose (HNSTD).
  • the present invention can design a single dose of the aza-T-dCyd drug to be lower than the value corresponding to the non-toxic level (NOAEL) or the highest dose that does not cause serious toxicity (HNSTD) based on Cmax rather than AUC, it is possible to expand the range of a very narrow therapeutic window of the aza-T-dCyd drug.
  • NOAEL non-toxic level
  • HNSTD serious toxicity
  • a database comparing in vitro data IC 50 , IC 60 , IC 70 , IC 80 and IC 90 with side effect values (IC 50 based on NOAEL and/or HNSTD) in a non-clinical large animal model can be constructed.
  • a single dose of the aza-T-dCyd drug is calculated from this, and then the maximum blood concentration (Cmax) of the aza-T-dCyd drug calculated therefrom is compared with the Cmax value corresponding to the non-toxic amount (NOAEL) or the highest dose that does not cause serious toxicity (HNSTD), and various therapeutic efficacy and/or side effect prediction information derived from a single dose of the aza-T-dCyd drug in a specific oral dosage form can be provided.
  • Cmax maximum blood concentration
  • NOAEL non-toxic amount
  • HNSTD serious toxicity
  • aza-T-dCyd has a low rate of degradation of cytidine deaminase compared to other cytidine analog-based anticancer drugs. Not only is the effect not significant, but the maximum blood concentration (Cmax) of the desired aza-T-dCyd drug can be maintained for a longer time there is
  • the present invention is characterized in that the ratio (wt%) of crystalline form A in the aza-T-dCyd drug is closely controlled to a known value within an error range, thereby solving problems caused by individual differences in absorption.
  • the present invention can provide an oral dosage form containing an aza-T-dCyd drug in which absorption of the drug and control of the maximum blood concentration (Cmax) are precisely controlled within an acceptable error range.
  • aza-T-dCyd is an analog of decitabine represented by Formula 1 and, like decitabine, can be prepared in various forms and crystal structures.
  • polymorphs of aza-T-dCyd include (pseudo-)polymorphs of aza-T-dCyd.
  • Polymorphism is a phenomenon in which one compound has more than one molecular arrangement structure, that is, a crystal structure, in a solid state, and has chemically identical but physically different characteristics.
  • the crystal structure means the internal structure of the crystal.
  • the most important characteristics of a solid are a specific distance between molecules and a specific bonding force, and polymorphs have different melting points because of the different distance and bonding strength between molecules, and therefore have different solubility.
  • Pseudo-polymorphism is chemically different and physically different. This is mainly in the case of solvates, in which solvent molecules enter the crystal lattice to form crystals.
  • the solvent is water, it is called a hydrate.
  • the synthesized product of the aza-T-dCyd compound mainly contains crystalline forms A and F, and may include various other crystalline forms (Example 1).
  • crystalline form A, crystalline form F, or a combination (mixed) form of crystalline form A and crystalline form F of aza-T-dCyd is disclosed.
  • Crystalline Form A of aza-T-dCyd can be defined as having peaks at 2 ⁇ diffraction angles of about 8o, about 13o, about 15o, about 17o, about 19o, about 22o, about 23o, about 26o, about 28o, about 29o, about 31o, about 33o, and about 37o in a powder X-ray diffraction spectrum.
  • crystalline form A of aza-T-dCyd has 2 ⁇ diffraction angles of 7.7° ⁇ 0.3°, 13.02° ⁇ 0.3°, 15.34° ⁇ 0.3°, 16.78° ⁇ 0.3°, 18.62° ⁇ 0.3°, 19.42° ⁇ 0.3°, 21.94° ⁇ 0.3°, and ⁇ 22.90°. .3°, 25.70° ⁇ 0.3°, 26.64° ⁇ 0.3°, 27.86° ⁇ 0.3°, 28.63° ⁇ 0.3°, 29.45° ⁇ 0.3°, 31.42° ⁇ 0.3°, 32.70° ⁇ 0.3°, 34.72 ⁇ 0.3, 35.97° ⁇ 0.3° and 37. It may have a peak of 46 ° ⁇ 0.3 °.
  • Crystalline form F of aza-T-dCyd has diffraction angles in 2 ⁇ of about 6o, about 12o, about 13o, about 14o, about 16o, about 18o, about 20o, about 21o, about 22o, about 26o, about 27o, about 29o, about 30o, about 33o, about 35o, about 36o in the powder X-ray diffraction spectrum. o, about 39o, and about 41o.
  • crystalline form F of aza-T-dCyd has 2 ⁇ diffraction angles of 6.06o ⁇ 0.3°, 12.10o ⁇ 0.3°, 13.02o ⁇ 0.3°, 14.38o ⁇ 0.3°, 15.94o ⁇ 0.3°, 17.50o ⁇ 0.3°, 19.62o ⁇ 0.3°, 21.18o ⁇ 0.3°, 22.34o ⁇ 0.3°, 26.18o ⁇ 0.3°, 27.42o ⁇ 0.3°, 28.50o ⁇ 0.3°, 29.90o ⁇ 0.3°, 32.66o ⁇ 0.3°, 35.02o ⁇ 0.3°, 36.30o ⁇ 0.3 °, 38.94o ⁇ 0.3 °, and 41.06o ⁇ 0.3 °.
  • the first step may be to convert the crystalline polymorph containing the solvate to a single crystalline Form A by removing the solvent.
  • an aza-T-dCyd drug consisting only of crystalline Form A can be provided.
  • the second step is to provide an oral dosage form containing an aza-T-dCyd drug that is precisely designed within an acceptable error range for a single dose that exhibits a desired therapeutic effect from the highest blood concentration (Cmax) of the aza-T-dCyd drug.
  • the amorphous form dissolves quickly and shows a quick effect, and the duration is short. In the case of the crystalline form, it dissolves slowly, so the effect appears slowly but the duration is long.
  • the physicochemical properties (physical properties) of the aza-T-dCyd drug directly affect formulation development. Since polymorphs differ in solubility and stability, polymorphs are very important pharmaceutically, and in particular, crystalline forms that are well soluble are preferred in terms of bioavailability. However, simply selecting a form that dissolves well does not end all problems, and it is necessary to study whether or not the form is converted. Therefore, the present invention was completed through polymorph screen and characterization of aza-T-dCyd in Examples 4 and 5.
  • crystalline form A and crystalline form F are stable anhydride forms in physical and chemical terms. Therefore, constructing the aza-T-dCyd drug raw material in crystalline form A and/or crystalline form F with high physicochemical stability not only has advantages such as storage stability and purity control during formulation development, but more importantly, the crystalline form can affect the biological activity of the drug.
  • the dosage of the aza-T-dCyd drug may vary within a range depending on the dosage form (crystal form) and route of administration.
  • an oral dosage form containing aza-T-dCyd may be designed to dissolve 90% or more of Form A and/or Form F in the stomach.
  • the maximum blood concentration (Cmax) and/or the area under the blood drug concentration-time curve (AUC) of the aza-T-dCyd drug may be adjusted by adjusting the composition ratio of Form A and/or Form F of the aza-T-dCyd drug.
  • Form A exhibits a high dissolution rate/solubility in strong acidic conditions and exhibits a consistent dissolution rate profile compared to Form F.
  • the present inventors prepared various crystalline forms of aza-T-dCyd and confirmed that the anticancer effect in crystalline form A was the most excellent. Therefore, it is a feature of the present invention to apply this to the design of an oral dosage form containing an Aza-T-dCyd drug whose efficacy is dependent on Cmax.
  • the desired Cmax value of the aza-T-dCyd drug can be stably achieved within an acceptable error range by adjusting the dose of Form A and/or the ratio of the aza-T-dCyd drug, and by applying this, the aza-T-dCyd drug can be formulated to exert a desired therapeutic effect by minimizing individual variation.
  • amorphous and highly water-soluble additives may be added and mixed thereto to improve solubility and increase bioavailability.
  • the additive include starch, lactose, PVP, and microcrystalline cellulose.
  • the oral dosage form of the present invention may contain a disintegrating carrier so that at least 80% of the aza-T-dCyd drug is dissolved in an acidic stomach.
  • the amorphous form has high solubility, which helps to increase the efficacy of the drug and show rapid action, but it is difficult to release the drug and control the blood concentration; no absorption of the aza-T-dCyd drug from the cecum (Example 12); Form A and Form F have high dissolution rate/solubility and similar dissolution rates at pH 5 (pH condition of the small intestine) and pH 1.2 (pH condition of the stomach).
  • the present invention provides (1) a single dose of aza-T-dCyd drug and (2) aza-T-d by controlling the ratio of Form A to aza-T-dCyd drug showing a consistent dissolution profile in the stomach, Despite the very narrow therapeutic window of Cyd drugs, high Cmax can be easily achieved within an acceptable margin of error.
  • the present invention designs an oral dosage form that is decomposed by gastric acid so that, for example, 90% or more of crystalline form A dissolves in the stomach, thereby absorbing most of the aza-T-dCyd drug at the beginning of the small intestine, thereby exhibiting the desired therapeutic effect with a lower single dose than other crystalline forms.
  • a high Cmax can be precisely controlled with significantly reduced potential for toxic side effects.
  • the present invention provides a single dose of the aza-T-dCyd drug and a crystalline form of the aza-T-dCyd drug to precisely control (i) the highest blood concentration (Cmax) of the drug Aza-T-dCyd that exerts the desired therapeutic effect, optionally (ii) the amount of the drug aza-T-dCyd that reaches the systemic circulation from the oral dosage form, and optionally (iii) the time it takes for the drug drug aza-T-dCyd to reach the systemic circulation. It is characterized by designing the ratio of A.
  • the ratio of crystalline form A in the aza-T-dCyd drug may be wt% or mole%, but is not limited thereto.
  • the present invention can design a single dose of aza-T-dCyd drug and the ratio of crystalline form A in aza-T-dCyd drug in order to easily control the maximum blood concentration (Cmax) after drug administration, which indicates whether the drug is sufficiently absorbed into the systemic circulation to produce a therapeutic response.
  • Cmax maximum blood concentration
  • the dosage of the aza-T-dCyd drug and the ratio (wt%) of the crystalline form A in the aza-T-dCyd drug that is, the amount of the drug reaching the systemic circulation from the oral dosage form and the time taken to reach the systemic circulation can be easily controlled by adjusting the dosage of the aza-T-dCyd drug and the dosage of the crystalline form A.
  • the present invention can easily control the amount of the drug reaching the systemic circulation from the oral formulation and the time taken to reach the systemic circulation by adjusting the dose of Form A or the ratio (wt%) of the aza-T-dCyd drug.
  • a crystalline raw material containing crystalline form A in a desired ratio (wt%) can be prepared from aza-T-dCyd crude materials, which are synthesized products of the aza-T-dCyd drug, and then formulated into an oral dosage form.
  • the present invention provides an oral dosage form in which the ratio (wt%) of crystalline Form A in the aza-T-dCyd drug is controlled, wherein the crystalline raw material containing Form A in a desired ratio (wt%) is prepared from crude materials of aza-T-dCyd, which is a synthetic product of the aza-T-dCyd drug, and then formulated into an oral dosage form.
  • preparing a crystalline raw material containing crystalline form A in a desired ratio (wt%) from aza-T-dCyd crude materials, which are the synthetic product of the aza-T-dCyd drug can be a process of controlling the desired ratio (wt%) of crystalline form A within a ⁇ 10% error range, preferably within a ⁇ 5% error range, and the desired ratio (wt%) of crystalline form A is within a ⁇ 10% error range, preferably ⁇ 5% error range. It may be a verification process.
  • the oral dosage form of the present invention is characterized in that a single dosage of the aza-T-dCyd drug is designed so that the dosage of the aza-T-dCyd drug contains more than the ratio of Form A corresponding to the inflection point of the Cmax phase at which the maximum blood concentration (Cmax) change value increases according to the change in the ratio of Form A in the same single dose of the aza-T-dCyd drug.
  • the Cmax inflection point considered in designing the ratio of Form A in the drug Aza-T-dCyd may appear when the ratio of Form A in the drug Aza-T-dCyd is 50% to 80%.
  • the ratio of crystalline Form A in the aza-T-dCyd drug may be 70% or more.
  • the oral dosage form can be designed such that the ratio of Form A in the aza-T-dCyd drug is 70% or more.
  • the ratio of crystalline Form A in the aza-T-dCyd drug in the oral dosage form may be 100%.
  • the bioavailability of the aza-T-dCyd drug whose therapeutic effect/toxic side effect depends on Cmax can be precisely controlled during oral administration.
  • the oral dosage form may be formulated.
  • cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancers include, but are not limited to, blood-borne tumors (eg, multiple myeloma, lymphoma, and leukemia) and solid tumors.
  • hematological cancers include non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute granulocytic leukemia, acute myelogenous leukemia, chronic myeloid leukemia, etc.
  • Non-limiting examples of solid cancers include gastric cancer, kidney cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, colon cancer, breast cancer, melanoma, and pancreatic cancer.
  • patient and “subject” refer to animals such as mammals.
  • the patient is a human.
  • the patient is a non-human animal, such as a dog, cat, livestock (eg, horse, pig or donkey), chimpanzee or monkey.
  • the anticancer effect or therapeutic effect of an anticancer agent may refer to an action that reduces the severity of cancer, reduces the size of a tumor, or delays or slows down the progression of cancer, which occurs while a patient is suffering from a specific cancer.
  • the anticancer effect of an anticancer agent may be Cell Viability (a change in the degree of cytotoxicity or the number of cells) of cancer cells after treatment with the anticancer agent in vitro and/or in vivo. For example, it can be confirmed indirectly through a drug response test through a cell line or a non-clinical animal model (xenograft). In addition, even in cancer patients, the anticancer effect of the anticancer agent can be directly confirmed, and related data can be derived and used as a database. In addition, when designing an anticancer drug dosage guideline, animal model PK parameters and/or toxicity profile may be considered in parallel.
  • the anticancer effect of an anticancer agent may be inferred from in-vitro data, such as the % maximum effect of the anticancer agent, such as IC 50 , IC 60 , IC 70 , IC 80 and IC 90 , and the highest blood concentration of the drug (Cmax) and / or blood drug concentration - It can also be confirmed in non-clinical animal models and clinical cancer patients through in-vivo data such as area under the time curve (AUC). .
  • Reactivity of an anticancer agent means clinical sensitivity in terms of anticancer effect.
  • Sensitivity and “susceptibility” when referring to treatment with an anti-cancer agent are relative terms that refer to the degree of effectiveness of a compound in alleviating or reducing the progression of the tumor or disease being treated.
  • an "effective patient's anticancer effect/response” can be, for example, a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more inhibition of a patient's response, as measured by any suitable means, such as gene expression, cell counts, assays, and the like.
  • the dose is the dose at which drug efficacy is expected.
  • the medicinal effect may be an anticancer effect.
  • the reactivity (anti-cancer effect) of an anti-cancer agent is the degree of response, and may be the % maximum effect of the anti-cancer agent, such as IC 50 , IC 60 , IC 70 , IC 80 and IC 90 , and a value that exhibits toxicity to normal cells (LC 50 ).
  • dosage forms for oral use can be formulated using a variety of formulation techniques known in the art.
  • it may include a biodegradable (hydrolyzable) polymeric carrier used to adhere to the oral mucosa. It is designed to slowly erode over a predetermined period of time, wherein drug delivery is provided essentially entirely.
  • Drug delivery in an oral dosage form avoids the weaknesses encountered with oral drug administration, such as slow absorption, degradation of the active agent by fluid present in the gastrointestinal tract and/or first pass and inactivation in the liver, as recognized by those skilled in the art.
  • biodegradable (hydrolysable) polymeric carriers it will be appreciated that virtually any such carrier may be used provided that the desired drug release profile is not compromised, and that the carrier is compatible with aza-T-dCyd and any other ingredient present in an oral dosage unit.
  • polymeric carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the oral mucosa.
  • polymeric carriers useful herein include acrylic acid polymers and co, eg, those known as “carbomers” (Carbopol ® (available from B.F. Goodrich) is one such polymer).
  • carbomers Carbopol ® (available from B.F. Goodrich) is one such polymer.
  • other ingredients that can be incorporated into an oral dosage form include disintegrants, diluents, binders, lubricants, flavoring agents, coloring agents, preservatives, and the like. In some embodiments, it may be in the form of a conventionally formulated tablet, lozenge, or gel for buccal or sublingual administration.
  • administration of the compound is continued at the physician's discretion when the patient's condition improves;
  • the dose of drug to be administered may be temporarily reduced or temporarily discontinued for some length of time (ie, a "holiday").
  • the length of the washout can vary between 2 days and 1 year, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days. , 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during the washout is 10%-100%, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100 contains %.
  • a maintenance dose is administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced as a function of symptoms, to a level at which improved disease, disorder or condition is maintained.
  • patients require intermittent treatment over a long period of time upon any recurrence of symptoms.
  • the amount of a given agent that will correspond to such amount will vary with factors of the subject or host in need of treatment such as the particular compound, severity of the disease, identity (e.g., body weight), but can nevertheless be routinely determined in a manner known in the art, depending on, for example, the particular agent to be administered, the route of administration, and the particulars surrounding the case involving the subject or host to be treated. In general, however, doses used for adult human treatment will typically range from 0.02-5000 mg/day, or about 1-1500 mg/day.
  • a single dose herein may be given as a single dose or in divided doses administered simultaneously, for example as 2, 3, 4 or more sub-doses.
  • oral formulations are unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate amounts of one or more compounds.
  • the unit dose is in the form of patches containing discrete amounts of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers may be used, in which case it is typical to include a preservative in the composition.
  • formulations for parenteral injection are presented in unit dosage form, including but not limited to ampoules, or in multi-dose containers, with an added preservative.
  • the concentration of the drug in the body must be maintained within a therapeutic range for a certain period of time or longer.
  • a drug is present in excess in the body, it exhibits toxicity, and when the amount is too small, the therapeutic effect does not appear. Therefore, the bioavailability of the aza-T-dCyd drug can be controlled by adjusting the polymorphism of the aza-T-dCyd drug at the crystalline level.
  • the present invention uses the polymorphism of the aza-T-dCyd crystalline form to control the release and absorption of the aza-T-dCyd drug, thereby reducing the side effects of the aza-T-dCyd drug that is Cmax dependent rather than the AUC and maximizing the efficacy of the aza-T-dCyd drug.
  • the present invention can provide an oral dosage form in which the ratio (wt%) of crystalline Form A among polymorphs of aza-T-dCyd drug is adjusted in order to achieve the highest blood concentration (Cmax) within a single dose of the aza-T-dCyd drug, which is related to toxicity in the body, which is a side effect, based on a single dose.
  • the dosage of the aza-T-dCyd drug and the ratio (wt%) of crystalline Form A in the Aza-T-dCyd drug can be precisely and easily controlled.
  • Figure 1 shows representative HT-XRPD and HR-XRPD patterns for aza-T-dCyd starting material (SM: aza-T-dCyd for which no specific crystallization conditions have yet been applied).
  • Figure 2 shows representative simulated XRPD and HR-XRPD of aza-T-dCyd crystalline Form A.
  • Figure 3 shows a representative TGMS analysis of aza-T-dCyd starting material (SM).
  • Figure 4 shows a representative DSC trace of aza-T-dCyd starting material (SM).
  • FIG. 5 shows representative simulated XRPD and HT-XRPD of aza-T-dCyd Form A after second cycling DSC.
  • Figure 6 shows the cycle DSC of aza-T-dCyd starting material (SM).
  • FIG. 7A and 7B show representative results of LCMS of aza-T-dCyd starting material (SM). Specifically, FIG. 7A shows a representative LC chromatogram of aza-T-dCyd starting material (SM). 7B shows a representative MS spectrum of aza-T-dCyd from liquid chromatography.
  • SM aza-T-dCyd starting material
  • FIGS 8A-C show representative results of LCMS of aza-T-dCyd starting material (SM) after forming a solution in water.
  • FIG. 8A shows an LC chromatogram of aza-T-dCyd formulated in water.
  • Figure 8b shows the MS spectrum of the impurity eluted at 3.8 minutes.
  • Figure 8c shows the MS spectrum of aza-T-dCyd eluted at 4.4 min.
  • Figure 9 presents representative data showing the chemical stability of aza-T-dCyd in various solutions.
  • FIG. 11 shows a representative XRPD pattern of Form A of aza-T-dCyd.
  • 13A-C show representative chemical analyzes of Form A.
  • 13A shows the TGMS analysis of Form A.
  • 13B shows the DSC analysis of Form A.
  • 13C shows the LCMS analysis of Form A.
  • 15A-C show representative chemical analyzes of Form F.
  • 15A shows the TGMS analysis of Form F.
  • 15B shows the DSC analysis of Form F.
  • 15C shows the LCMS analysis of Form F.
  • 16 shows a representative XRPD pattern of Form F of aza-T-dCyd.
  • 17 and 18 present in vivo luciferase activity data showing tumor size when aza-T-dCyd starting material (SM) was administered to female NOD-SCID mice.
  • SM aza-T-dCyd starting material
  • Fig. 19 shows the half-maximal inhibitory concentration (IC50) when hematological malignant cells (Mv4-11) were treated with aza-T-dCyd starting material (SM).
  • Figure 23 shows IC 50 values when the K562 cell line was treated with crystalline Form A or SM.
  • Figure 24 shows the IC 50 values when the HL-60 cell line was treated with Form A or SM.
  • 25 shows PK analysis results obtained by blood sampling at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hr after oral administration by adjusting the ratio (wt%) of crystalline form A in Aza-T-dCyd drug.
  • 26A and B show dissolution rate profiles of PO and IC administration of 1mpk and 3mpk of aza-T-dCyd compound, respectively.
  • aza-T-dCyd was prepared and analyzed by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetry-mass spectrometry (TGMS), and liquid chromatography/mass spectrometry (LCMS).
  • the starting material (SM) is aza-T-dCyd that has not yet been subjected to specific crystallization conditions.
  • Figure 1 shows high-throughput XRPD (HT-XRPD) and high-resolution XRPD (HR-XRPD) in the top and bottom patterns, respectively.
  • the starting materials include crystals suitable for single crystal structure analysis.
  • crystalline form A As a result of crystallization of the starting material, a crystalline form having an asymmetric monoclinic P21 space group was obtained, which is referred to as crystalline form A.
  • Table 1 provides the relevant dimensions of Form A.
  • the HR-XRPD pattern of the starting material was compared with the HR-XRPD pattern, which is a simulated pattern of a single crystal of crystalline Form A, and is shown in FIG. 2 .
  • the crystal A has peaks in 7.7 °, 13.02 °, 15.34 °, 16.78 °, 18.62 °, 19.42 °, 21.94 °, 22.90 °, 25.70 °, 27.86 °, 28.70 °, 31.42 °, 32.70 °, and 37.46 ° 2 ⁇ . Based on this comparison, the starting material is calculated to contain about 70% of crystalline Form A and about 30% of the other crystalline forms of aza-T-dCyd.
  • TGMS analysis of the starting material between 25-300 °C (10 °C/min) showed a mass loss of 11.7% between 100-170 °C, most likely due to organic solvents (Fig. 3). Simultaneously with the mass loss, the heat flow signal showed two endotherms with an exotherm in between. A third endotherm was observed around 195 °C due to the onset of melting and decomposition.
  • XRPD and single crystal structure analysis revealed that the starting material consisted of a mixture of crystalline phases.
  • two cyclic DSC experiments were performed on the starting materials. One sample was heated to 170°C and cooled back to room temperature. As a result of analyzing the obtained solid by XRPD, it was consistent with the simulated pattern of crystalline Form A (FIG. 5).
  • the starting material was heated to 170 °C, cooled to 25 °C and then heated to 300 °C (Fig. 6). No thermal phenomena were observed during cooling, and only endothermic melting was observed at 194 °C in the second heating cycle, confirming the melting temperature of Form A.
  • an amorphous material was generated from the starting material via a lyophilized solution method of aza-T-dCyd.
  • Water, water/1,4-dioxane (50/50), water/THF (50/50) and water/tert-butyl alcohol (50/50% (v/v)) were added to aza-T-dCyd to obtain a solution of aza-T-dCyd in organic solvent for freeze-drying experiments. Lyophilization of the aza-T-dCyd solution resulted in poor crystalline materials containing impurities.
  • thermodynamic solubility of aza-T-dCyd was determined according to the shake flask method. A suspension of crystalline aza-T-dCyd was prepared in 25 pure solvents. A small amount of solvent was added to aza-T-dCyd until thin suspensions were obtained. The sample was then equilibrated at room temperature under continuous stirring for 24 hours. After equilibration, a small amount of mother liquor was filtered and analyzed by HPLC. Concentrations of solutes were determined against a calibration curve of aza-T-dCyd. The solubility values of aza-T-dCyd at room temperature are listed in Table 3 according to the United States Pharmacopoeia classification (USP29).
  • aza-T-dCyd was dissolved in high-boiling solvents such as DMF and DMA.
  • high-boiling solvents such as DMF and DMA.
  • aza-T-dCyd is slightly or very slightly soluble in polar solvents and sparingly soluble in non-polar solvents.
  • a polymorph screen was performed with various pure organic solvents and solvent mixtures of various compositions, combining six different crystallization methods.
  • the screening experimental conditions were chosen as follows: (1) the experiment was started with a crystalline starting material; (2) the compound stayed in solution for a limited time ( ⁇ 5 days); (3) avoided high temperatures ( ⁇ 50 °C); (4) solid aza-T-dCyd is handled in a glovebox under as dry conditions as possible (relative humidity about 20%) to avoid moisture absorption; (5) avoided water and limited ketone use; and (6) moderate stress conditions to evaluate the physical stability of the resulting solid.
  • Solvent equilibration experiments were performed at two temperatures: RT for 1 day and 5 °C for 5 days. Suspensions of aza-T-dCyd were prepared in different solvents along with the crystalline starting material and the solid was separated from the mother liquor upon completion of the equilibration time.
  • Anti-solvent experiments were performed using 10 solvent and anti-solvent combinations by reverse addition. A small amount of a highly concentrated solution of aza-T-dCyd was added to 20 mL of anti-solvent (Step 1).
  • Thermocycling experiments were performed by preparing aza-T-dCyd suspensions in various solvents and solvent mixtures at room temperature. The resulting suspension was subjected to a temperature profile of 5 to 50 °C.
  • Sonication experiments were performed by sonicating crystalline starting materials in the presence of small amounts of solvent.
  • Vapor diffusion experiments into solution were performed with the slow method of anti-solvent crystallization.
  • a saturated aza-T-dCyd solution was exposed to anti-solvent vapor for one week at room temperature.
  • Form A was the most abundant crystalline phase recovered from the screening experiment. Form A was found in all crystallization methods and in various solvents and solvent mixtures. From solvent equilibrium experiments, it was observed that Form A was obtained as a pure phase from solvents in which aza-T-dCyd was slightly or very slightly soluble.
  • the peak at 26.3° 2 ⁇ belonged to Form B.
  • the observed peak at 16.0° 2 ⁇ represents Form C1 and the peaks at 16.0 and 17.6° 2 ⁇ were attributed to Form C2.
  • the observed peak at 24.8° 2 ⁇ is attributed to Form D1 and the peaks at 24.8 and 34.1° 2 ⁇ are attributed to Form D2. According to this assignment, some solids were classified as crystalline forms A+D1/D2, A+C1/C2 or A+B+D2.
  • Form B was obtained as a pure phase by solvent equilibration in DMA and DMF both at room temperature and 5° C. and also from thermocycling experiments in DMSO/2-ethyl-1-hexanol (50/50). Form B was physically unstable and converted to Form A after storage at 25oC and 60% relative humidity.
  • Classes C and D were not observed as pure crystalline phases but were always admixed with Form A. In most cases, these mixtures converted to Form A after storage at 25° C. and 60% relative humidity.
  • Form F was obtained from vapor diffusion or evaporative crystallization in various solvents. Form F was physically stable. The peaks of Form F are 6.06°, 12.10°, 13.02°, 14.38°, 15.94°, 17.50°, 19.62°, 21.18°, 22.34°, 26.18°, 27.42°, 28.50°, 29.90°, 32.66°, 35.0 2°, 36.30°, 38.94°, and 41.06° 2 ⁇ .
  • Crystalline forms G1 and G2 have similar XRPD patterns, where some peaks shift between the two forms.
  • Form G1 was obtained from avoiding solvent addition or from sonication.
  • Form G2 was obtained from evaporative crystallization using DMA/EtOH. Both Form G1 and Form G2 were converted to Form A after storage at 25°C and 60% relative humidity.
  • Form H was obtained from evaporative crystallization in several solvent mixtures. This form is unstable. When obtained from NMP, Form H was converted to Form F. Form H was converted to Form A when obtained from other solvents.
  • Form I was obtained from evaporative crystallization from DMSO/IPA. Form I was converted to Form A after storage at 25°C and 60% relative humidity.
  • Form J was obtained from vapor diffusion into a solution with DMF as solvent and THF as anti-solvent. Form J was converted to Form A after storage at 25°C and 60% relative humidity.
  • Form K was observed in mixtures with Form F after evaporative crystallization from DMF. Form K was converted to form F after storage at 25°C and 60% relative humidity.
  • Form L was observed in the solid after storage at 25° C. and 65% relative humidity.
  • the XRPD patterns for each of these new forms are shown in FIG. 12 .
  • Form A obtained from solvent equilibration experiments of RT in TFE was used for analytical characterization.
  • TGMS results showed residual solvent release of about 0.7% in the temperature range of 30 - 190 °C (FIG. 12A).
  • An endotherm was observed in the DSC trace at 205 °C due to melting and decomposition (Fig. 12B).
  • LCMS analysis confirmed the integrity of 100% (area %) purity of Form A (FIG. 12C).
  • Form F obtained from evaporative crystallization experiments using DMF/acetonitrile (80/20, v/v) was used for characterization.
  • the TGMS results showed a small loss of 1.1% between 30 and 140 °C, which may be mostly due to residual solvent (FIG. 15A).
  • the DSC trace showed one endothermic event at about 170 °C due to melting and decomposition (Fig. 15B).
  • LCMS analysis confirmed the integrity of the API with 100% purity (area %) (FIG. 15C).
  • Form A has a higher melting temperature than Form F and can be considered a more thermodynamically stable form. Both crystalline forms A and F are anhydrous.
  • Forms B, C2, D2, E, G1, G2, H, I, J and K convert to Form A when solvated and stored at 25° C., 60% relative humidity for 2 days.
  • Form B obtained from solvent equilibration experiments in DMA at room temperature was further characterized.
  • the TGMS results showed a gradual mass loss upon heating between 30 and 170 °C with a mass loss of 25.0%.
  • the temperature at which decomposition begins is not clear due to the gradual loss of mass on heating.
  • Form B can be a non-stoichiometric solvate that can be formed with different solvents.
  • LCMS analysis indicated a solid purity of 97.3% aza-T-dCyd and the presence of impurities of 2.7% (area %).
  • Form C2 showed two additional peaks observed in the XRPD pattern in mixtures with other forms. TGMS analysis showed a mass loss of 0.7% over the temperature range of 30 - 160 °C. The heat flow signal showed only one endothermic event around 190 °C, which could be related to the melting and decomposition of Form A. The investigation of form C2 is inconclusive because form C2 is only present in trace amounts in mixtures with crystalline form A. Therefore, the nature of this form is still unclear. However, it appears to be a true (pseudo-)polymorph of aza-T-dCyd, as the chemical purity of the total solid sample was 100% (area %).
  • Form D2 exhibited two additional peaks observed in the XRPD pattern in mixtures with Form A. TGMS analysis of Form A+D2 showed that Form D2 was most likely the solvated form. A mass loss of 5.1% was observed between 90 and 170 °C. The results were inconclusive with respect to the released solvent. LCMS analysis of the crystalline mixture confirmed the integrity of aza-T-dCyd with a chemical purity of 100% (area %).
  • Form E from evaporative crystallization experiments using DMA was further analyzed by TGMS and LCMS.
  • the TGMS results showed a 25.8% mass loss of DMA, which corresponds to 1 molar equivalent of solvent.
  • the solvent was released in a stepwise manner between 90 and 160 °C, suggesting that Form E is a mono-DMA solvate.
  • an endothermic event was recorded at 200 °C, probably corresponding to melting of Form A. Compound integrity was confirmed by LCMS analysis.
  • Class G is an isostructural class of solvates. Crystalline forms G1 and G2 were further characterized by TGMS and LCMS. LCMS analysis confirmed compound integrity (area % of 100%). Form G1 obtained from antisolvent addition experiments using NMP and cyclohexane was used for characterization. TGMS results showed a stepwise mass loss of 27.5% between 90 and 160 °C. The 27.5% mass loss corresponds to approximately one molecule of NMP per molecule of aza-T-dCyd, so Form G1 may be a mono-NMP solvate. The DSC signal recorded two endotherms around 110 and 150 °C due to solvent loss and a third endotherm at 200 °C, which may correspond to the melting of Form A.
  • Form G2 was obtained by evaporative crystallization in DMA/ethanol (80/20, v/v).
  • the 14.6% mass loss observed by TGMS between 70 and 120 °C corresponds to 0.5 molar equivalents of DMA.
  • two endotherms were observed around 80 and 90 °C due to solvent loss, and a third endotherm around 195 °C due to melting and decomposition.
  • Form H obtained from evaporative crystallization from NMP/THF (80/20, v/v) was used for characterization of Form H.
  • the gradual mass loss observed by TGMS analysis was 15.3% from 30 to 180°.
  • C corresponds to about 0.5 molar equivalent of NMP.
  • an extensive endotherm was observed around 130 °C.
  • Form H was observed in experiments with other solvents and is therefore most likely a non-stoichiometric solvate capable of incorporating other solvent molecules into the crystal structure.
  • a second extensive endotherm was observed in the DSC trace due to decomposition at around 220 °C. From the TGMS data, it is not clear where solvent loss ends and where thermal decomposition begins. Events may partially overlap. To obtain dry samples, the solids had to be vacuum dried at 50 °C for 24 hours. This may have affected the purity as the LCMS data indicated that the solid was 82% (area %) pure.
  • Form I was obtained by evaporative crystallization from DMSO/IPA (80/20, v/v). TGMS data showed a gradual mass loss of 14.7% between 30 and 170 °C. A mass loss of 14.7% corresponds to about 0.5 molar equivalent of DMSO. Form I may be a hemi-DMSO solvate. The DSC trace showed two broad endotherms at 70oC and 110 °C due to mass loss, and a third endotherm around 190 °C due to melting and decomposition processes.
  • Form J precipitated by vapor diffusion from solution using DMF and THF, was further characterized.
  • TGMS data showed a 7.6% mass loss of THF stepwise between 120 and 170 °C.
  • the mass loss corresponds to about 0.3 molar equivalents of THF and Form J is therefore most likely a non-stoichiometric solvate.
  • the DSC trace recorded two endotherms at 120 and 150 °C due to solvent loss, and the third endotherm recorded at 200 °C, consistent with the melting/decomposition event of Form A.
  • Form K was observed once in a mixture with Form F and was obtained by evaporation from a DMF solution. The mixture was further characterized. TGMS analysis showed a mass loss of 6.3% between 30 and 160 °C, probably due to the loss of DMF. The mass loss was accompanied by a small endotherm around 110 °C. Two large endotherms were observed at 180 and 195 °C. The endotherm at 195 °C may be due to melting and decomposition of Form A. Since Form K is a mixture with Form F (the unsolvated form), Form K is most likely the solvated form.
  • Form L was a poor crystalline solid observed only after storage at 25° C., 60% relative humidity and very low yield. TGMS analysis observed a mass loss of 2.8% between 30 and 170 °C followed by decomposition. The absence of thermal events in the DSC traces may be due to the small amount of sample used for analysis. It is not clear whether the mass loss is due to solvent trapped in the crystal structure or residual solvent. The nature of Form L is unclear as no further characterization could be performed.
  • aza-T-dCyd starting material; SM; aza-T-dCyd not yet subjected to specific crystallization conditions
  • Aza-T-dCyd starting material was administered to 6 female NOD-SCID mice divided into 4 groups.
  • Group 1 was the vehicle control group.
  • Group 2 was administered with 2.0 mg/kg of aza-T-dCyd starting material (SM) once a day, and group 3 was administered with 1.0 mg/kg of aza-T-dCyd starting material (SM) twice a day.
  • the aza-T-dCyd starting material (SM) was administered in the above amount for 5 days, followed by a 2-day break, another 5 days of administration, and then a 9-day break. This cycle repeated itself.
  • SM aza-T-dCyd starting material
  • tumor size increased in group 1 (vehicle control group).
  • group 1 vehicle control group
  • the increase in tumor size was most significantly suppressed in group 2.
  • group 2 the increase in tumor size was most significantly suppressed in group 2.
  • group 3 the size of the tumor rapidly increased after 40 days of administration. From this, it was found that aza-T-dCyd was Cmax dependent rather than AUC dependent.
  • the tumor size of group 2 (2.0 mg/kg, once a day) was significantly smaller than that of group 1 (1.0 mg/kg, twice a day), demonstrating the results on day 43.
  • hematological malignant cells (Mv4-11) were treated with aza-T-dCyd starting material (SM), and the half-maximal inhibitory concentration (IC 50 ) was measured at 1 hour, 2 hours, and 4 hours.
  • the results are shown in FIG. 19 .
  • the measured IC 50 at 1 hour was about 160 nM, so the IC 50 at 2 hours was expected to be 80 nM and the IC 50 at 4 hours to be 20 nM.
  • the IC 50 measured at 2 h was about 120 nM, much higher than the expected value of 80 nM.
  • the IC 50 measured at 4 hours was about 80 nM, much higher than the expected value of 20 nM.
  • SM aza-T-dCyd starting material
  • the data suggest that crystalline polymorphs with large dissolution profiles, such as Form A or Form F in Example 7 described below, are superior to aza-T-dCyd starting material (SM) and other crystalline polymorphs with inferior dissolution profiles. Also for the same reason, the data suggest that crystalline polymorphs such as Form A or Form F exhibit improved PK profiles over the aza-T-dCyd starting material (SM) and other crystalline polymorphs.
  • SM aza-T-dCyd starting material
  • the crystalline form usually has disadvantages in solubility compared to the amorphous form, in the case of crystalline forms A and F, although they are physicochemically stable forms, the dissolution characteristics according to the pH condition are very uniform and rapidly. Since they dissolve stably, the crystalline form can be controlled and used according to the need for formulation development.
  • Example 7 Dissolution Profiles of Form A and Form F at Various pH Points
  • Form A and Form F exhibited similar dissolution rates, whereas Form A exhibited a more consistent dissolution profile compared to Form F.
  • pH 6.5 and pH 5 pH conditions of the appendix and small intestine
  • Form A can be prepared into a variety of drug forms that target release of the drug's active ingredient at about pH 1.2 (e.g., stomach or large intestine). Further, this suggests that Form F can be made into various drug forms that target release of the drug's active ingredient at about pH 5.0-6.5 (eg, small intestine).
  • the aza-T-dCyd starting material (SM), crystalline form A, and crystalline form F were prepared in the form of capsules mixed with microcrystalline cellulose in an 8:92 (w/w) ratio, respectively, at 2 mg/kg of SM, crystalline form A or crystalline form F.
  • Each of the SM capsules, crystalline A capsule, and crystalline Form F capsule was administered at a dose of 2 mg/kg to 2 male SD rats (i.e., a total of 6 male SD rats).
  • plasma concentrations of each of SM, Form A and Form F in tested SD rats were measured at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after capsule administration.
  • Form A and Form F showed higher Cmax values than SM.
  • Form A showed a Cmax value about 1.3 times higher than SM
  • Form F showed a Cmax value about 1.4 times higher than SM.
  • both crystalline form A and crystalline form B showed about 30% higher AUC values than SM.
  • Example 9 AZA-T-DCYD Comparison of half maximal inhibitory concentration (IC 50 ) of starting material and Form A
  • K562 and HL-60 cell lines were cultured and maintained in RPMI (10% FBS, 1% penicillin-streptomycin) medium at 37°C, 95% air and 5% CO 2 .
  • K562 and HL-60 cell lines were each seeded in 96-well plates at a density of 3000 cells/well (90 ⁇ l). Crystalline A and SM were treated in 10 ⁇ l using a 3-fold dilution, and each well was treated at a final concentration of 10 ⁇ M. Cells were incubated for 3 days at 37°C, 95% air and 5% CO 2 . The 96-well plate was left at room temperature for 30 minutes to equilibrate.
  • Form A exhibits an IC 50 value about 5% lower than SM, providing a greater effect.
  • Example 10 PK experiment in rats according to the ratio (wt%) of crystalline form A in drug Aza-T-dCyd
  • samples for PK experiments upon oral administration were prepared as follows.
  • a drug raw material of Aza-T-dCyd with 100% pure crystalline form A was prepared.
  • pure crystalline Form F could not be obtained, and the obtained Aza-T-dCyd drug substance was a polycrystalline form composed of 52% of crystalline form F, 15% of crystalline form A, and 33% of other undefined forms.
  • This polycrystalline Aza-T-dCyd drug substance was named polycrystalline form F'.
  • the experimental group for the PK experiment consisted of a total of 11 groups, and the ratio (mole %) of Aza-T-dCyd in each experimental group (crystal form A: other forms) of Aza-T-dCyd is shown in Table 13 below.
  • PK analysis was performed by blood sampling at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hr after oral administration, and the results are shown in Table 14 and FIG. 25.
  • Aza-T-dCyd significantly increased Cmax to 852-897 ng/mL when the ratio of crystalline form A was 70% or more. Specifically, when the ratio of crystalline form A in the Aza-T-dCyd drug is 70% or more in rats, the Cmax stably shows 850 ng/mL, but when the ratio is less than 70%, the Cmax has 442-703 ng/mL.
  • mice with 2mpk and 1mpk twice As described above, when comparing the results of treating mice with 2mpk and 1mpk twice, the weight loss of mice was severe in the latter. In addition, when the in vivo luciferase activity was measured, when the treatment was divided into two times, the rate of increase was steeper, so it can be predicted that the tumor inhibitory effect is more excellent when 2mpk is treated at one time.
  • a desired therapeutic effect can be exerted by sufficiently exhibiting the drug effect through rapid treatment in a short period of time.
  • the present invention can solve the problem that the variance in drug exposure among individual patients is very high, so that the optimal administration dose for one individual becomes a dose that cannot show a therapeutic effect for another individual, and for another individual, a dose that causes severe toxicity, resulting in a very narrow therapeutic window.
  • Example 12 Absorption profile in the gastrointestinal tract when formulated for oral dosage form
  • the Aza-T-dCyd drug was orally administered (PO) to the mice, and after inserting the Aza-T-dCyd drug into the caecum with a cannula, plasma concentration graphs as shown in FIG. 26 were obtained.
  • 26A and 26B show plasma concentration distribution graphs showing drug absorption through the gastrointestinal tract in the case of PO as a result of PO and IC administration of 1mpk and 3mpk, respectively.
  • IC a very small amount of drug appears to be present in the plasma, that is, it shows a very low exposure to plasma compared to PO. That is, when administered orally, Aza-T-dCyd is not absorbed from the caecum (the digestive organ that swells like a pouch at the beginning of the large intestine).
  • the aza-T-dCyd drug is not absorbed in the postcecal region, which is the starting point of the large intestine, toxic side effects can be controlled in a narrow treatment window of the aza-T-dCyd drug.
  • Cmax can be implemented within the therapeutic window range, and toxic side effects due to the narrow therapeutic window can also be solved.

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Abstract

La présente invention concerne une formulation orale contenant de la 5-aza-4'-thio-2'-désoxycytidine et son procédé de préparation. La présente invention est destinée à résoudre le problème de disparités importantes parmi des patients quant à l'efficacité de médicament ou à l'exposition au médicament par rapport à un médicament anticancéreux à base de cytidine tel que la décitabine.
PCT/KR2023/001028 2022-01-21 2023-01-20 Formulation orale contenant de la 5-aza-4'-thio-2'-désoxycytidine et son procédé de préparation WO2023140691A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006037024A2 (fr) * 2004-09-27 2006-04-06 Supergen, Inc. Sels de decitabine
KR20140088603A (ko) * 2011-11-01 2014-07-10 셀진 코포레이션 시티딘 유사체의 경구 제제를 사용하여 암을 치료하는 방법
KR20170054997A (ko) * 2015-11-09 2017-05-18 서울대학교산학협력단 데시타빈 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골수유래억제세포 저해용 조성물

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Publication number Priority date Publication date Assignee Title
WO2006037024A2 (fr) * 2004-09-27 2006-04-06 Supergen, Inc. Sels de decitabine
KR20140088603A (ko) * 2011-11-01 2014-07-10 셀진 코포레이션 시티딘 유사체의 경구 제제를 사용하여 암을 치료하는 방법
KR20170054997A (ko) * 2015-11-09 2017-05-18 서울대학교산학협력단 데시타빈 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골수유래억제세포 저해용 조성물

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JAIDEEP V. THOTTASSERY, VIJAYA SAMBANDAM, PAULA W. ALLAN, JOSEPH A. MADDRY, YULIA Y. MAXUITENKO, KAMAL TIWARI, MELINDA HOLLINGSHEA: "Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine", CANCER CHEMOTHERAPY AND PHARMACOLOGY, SPRINGER VERLAG , BERLIN, DE, vol. 74, no. 2, 1 August 2014 (2014-08-01), DE , pages 291 - 302, XP055643162, ISSN: 0344-5704, DOI: 10.1007/s00280-014-2503-z *
SOOK HONG: "Pinotbio, Inc.'s DNMT1 Inhibition Targeted Anti-cancer Drugs Candidate, Disease Control Rate 78.6%", HIT NEWS, 9 June 2021 (2021-06-09), XP093078245, Retrieved from the Internet <URL: http://www.hitnews.co.kr/news/articleView.html?idxno=34533> [retrieved on 20230902] *

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